The
green fluorescent protein (GFP) has become an extremely
exciting and useful marker for gene expression. This
note describes a method to assay whole living E. coli
cells for the presence of GFP using the Turner BioSystems
TD-700 Laboratory Fluorometer. It will arrive at an
estimate of mature GFP in viable cells collected from
liquid broth.

This
is different from quantitating GFP in cell extracts
and at various stages of purification. One of the
problems encountered when measuring whole cell suspensions
is that such solutions are extremely turbid. This
has the effect of scattering the excitation light
in the solution thereby reducing the amount of light
reaching the GFP chromophore. In addition, light scattering
may contribute to false emission readings. To overcome
these problems, we calibrated the TD-700 against a
GFP concentration curve in cell solutions that do
not produce GFP, and in solutions prepared and read
in a frosted 13mm x 100mm borosilicate cuvette. One
of the drawbacks with measuring GFP in whole cells
as it is being produced is that its chromophore forms
slowly in the presence of molecular oxygen (O2). As
a consequence, any direct measurement is probably
an estimate of the total GFP present since a portion
of the GFP in whole cells has yet to become fluorescent.

Frosted
Cuvette: The lower 3/4 of a 13mm x 100mm borosilicate
test tube is sanded thoroughly on the outside with
#60 Aluminum oxide sand paper (dry), followed by a
thorough sanding with #320 silicone dioxide sand paper
(wet), to achieve a frosted effect.

3.
Fluorometer Calibration

3.1
Set-up

Upon
receiving and unpacking your TD-700, prepare it for
GFP quantitation.

Follow
the procedure outlined in your operation manual
to install the near UV mercury vapor lamp.

Install
your GFP filters into the filter cylinder and
place it into the fluorometer (remember to wipe
any fingerprints off of any filter or cuvette
after you handle them). The ports for each set
of filters are labeled EX for excitation and EM
for emission and each pair of ports is labeled
A through D. Choosing one pair of ports, carefully
insert your filters. The 390 nm filter has a reflective
face which should be installed so that it will
face out towards the lamp. Each filter is held
in place by a circular rubber grommet, or o-ring.

At
the ends of the filter cylinder are labeled marks
corresponding to the pair of filter ports you
have chosen. Insert the filter cylinder into the
fluorometer while aligning this mark with the
silver alignment mark found on the inside rim
of the fluorometer's sample chamber.

3.2
Calibration

Close
the TD-700 lid and turn the unit on. It will count
down 600 seconds to warm up.

After
the instrument warms up, insert the cuvette holder
into the sample chamber. Note that the top of
the cuvette holder has an arrow shape molded to
the top part of the holder. Orient this arrow
pointing toward the silver alignment mark on the
inside rim of the sample chamber.

You
will be performing a multi-optional mode calibration
(refer to page 21 in your manual if needed). Press
[enter] on the keypad. Enter [1] on the keypad
to enter SETUP, then press [1] again to enter
MODE. Using the ARROW key to choose the mode,
select MULTI-OPTIONAL then press [ESC] to return
to the setup menu.

Enter
[3] to enter the units menu, then use the ARROW
key to select µg/mL. Press [enter] to return to
the setup menu.

Enter
[2] to enter the calibration procedure menu, and
select DIRECT CONCENTRATION using the ARROW key.
Press [enter], then [ESC].

Enter
[2]. Enter the Max Range you want to use (the
default range, 999.9, is fine). Enter [1] to select
the range given, or [9] to change it. When you
select [1], you will be queried for the number
of standards you will be using (between 1 and
5). The suggested dilution range for this method
is 5 µg/ml to 1 µg/ml (i.e. five standards) in
buffer using a frosted cuvette or in a non-GFP
expressing cell suspension, in PBS, in an unfrosted
cuvette. Enter the number of standards you will
be using, then press [enter] and insert the first
sample to be read. Make sure the volume in the
tube is over 3 ml, so it is above the window of
the cuvette holder.

The
fluorometer will ask you if the concentration
value is correct. If it is, enter [1], if it is
not, enter [9] and input the correct value. When
you select [1], the machine will prompt you to
select the [*] button to store that sample into
memory. The cycle will repeat as it asks you for
the next highest standard.

When
you finish calibrating the lowest standard in
your series, you will be prompted for your blank,
which will be your cell suspension or buffer solution
without any GFP. Place that in the TD-700, and
press [enter]. The TD-700 will zero itself. When
the value at the right of the display becomes
stable, enter [0] as requested. It will record
your blank and indicate that the calibration is
complete.

The
TD-700 will give you a direct concentration value
when you insert your samples and close the chamber
door.

4.
Quantitating GFP In Intact Bacteria

4.1
Place the sample of E. coli suspension
to be assayed for GFP into a 13 mm x 100 mm borosilicate
test tube (frosted or unfrosted). If your cells were
cultured in media possessing residual autofluorescence,
such as LB broth, you can remove it by pelleting the
cells using centrifugation and resuspending them in
PBS. Always make sure the final volume in the tube
is over 3 ml (as the meniscus of the sample will affect
your readings).

You
have a strain of E. coli expressing GFP
under the control of the lac promoter which was
grown in a 55 ml liquid culture under induction
overnight. Simultaneously prepare your standard
solutions by growing and preparing untransformed
E. coli under the same conditions.

Pellet
50 ml of the cell suspension and wash several
times with 50 ml PBS. Resuspend the cells in 5
ml PBS (which will increase our readings 10-fold).

Perform
the above mentioned calibration and quantitation
procedure (sections 3 and 4) to estimate the concentration
of GFP in your transformed cells.

Comparison
of typical GFP concentration estimates in this
example for frosted and unfrosted cuvettes, after
considering your 10-fold increase in readings:

These
numbers are very similar and reflect an estimation
of the concentration of mature GFP present in these
cells. They also indicate that using a frosted cuvette
instead of suspended cells to calibrate the fluorometer
might be a workable alternative when you need to
quickly estimate the amount of GFP that is present
in your cells.

This
GFP application note was written by Daniel G. Gonzalez
M.S., who is currently a Biochemistry Ph.D candidate
working in the laboratory of William W. Ward at Rutgers
University (New Brunswick, N.J.). His interests include
the physical characterization of chromophore formation
in a variety of green fluorescent proteins from various
organisms. He was assisted by John Covalesky, who
has recently graduated from Rutgers University with
a B.S. in Biochemistry. William W. Ward is one of
the pioneers in GFP research and is still very active
in the field. He and Daniel currently use a wide assortment
of techniques in fluorescence analysis, protein purification
and molecular biology to study and characterize green
fluorescent Proteins. The Ward lab prepares and coordinates
a series of short courses in biotechnology that features
Aequorea GFP as a model protein for purification and
molecular manipulation. Information on these courses
can be obtained by contacting Daniel: